Rt2 pcr arraydataanalysisquickcarde

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Rt2 pcr arraydataanalysisquickcarde

  1. 1. Quick Reference: RT2 Profiler PCR Array Data Analysis RT2 Profiler PCR Array Data Analysis v3.5 http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php revision date 9 November 2012 Overview The RT2 Profiler PCR Array Data Analysis Quick Reference Card contains instructions for analyzing the data returned from using qRT-PCR Assays or RT2 Profiler PCR Arrays. For more information on the bench procedure for these products, please refer to the appropriate user manual or handbook. If you require additional assistance, please contact your local QIAGEN technical representative. Important Notes: • • • Please note that you must complete all of your work with the Data Analysis Web Portal in the same session. The data is not stored on a server, so all work is lost once the session (or the web browser) is closed. Be sure to export all processed data and results to an Excel file saved on your local computer. Please set the screen resolution to 1024 × 768 or greater, if possible. Turn off any window pop-up blockers. The software will launch separate windows for viewing the plots and charts. Setting baseline and threshold setting on the real-time thermocycler: The RT2 Profiler PCR Array Data Analysis Webportal analyzes CT values to calculate changes in gene expression. The CT values must be exported from your qPCR instrument and formatted into an Excel spreadsheet. All of the plates/wells must be copied into a single worksheet. 1. Calculate the threshold cycle (CT) for each well using the real-time cycler software, as described in the following steps. Note: If using the Roche LightCycler 480, there are 2 options for data analysis: using the second derivate max setting (in this case there is no need to calculate the CT) or using “Fit Points” (in this case the CT should be defined manually as described in step 3). 2. Define the baseline by choosing the automated baseline option if the cycler has the adaptive baseline function. If the cycler does not have the adaptive baseline function, set the baseline manually. To set the baseline manually, use the linear view of the amplification plots to determine the earliest visible amplification. Set the cycler to use the readings from cycle number 2 through 2 cycles before the earliest visible amplification, but no more than cycle 15. The earliest amplification will usually be visible between cycles 14 and 18. 3. Manually define the threshold by using the log view of the amplification plots. Choose a threshold value above the background signal but within the lower one-third to lower one-half of the linear phase of the amplification plot. Note: Ensure that the threshold values are the same across all RT2 Profiler PCR Array runs in the same analysis. The absolute position of the threshold is less important than its consistent position across arrays. If the RNA sample is of sufficient quality, the cycling program has been carried out correctly, and threshold values have been defined correctly, the value of CT PPC should be 20±2 for all arrays or samples. 4. Export or copy the CT values from each experimental run into an Excel Spreadsheet. CT values for each sample should be in a single column. Page 1 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu
  2. 2. Quick Reference: RT2 Profiler PCR Array Data Analysis Formatting data for the Data Analysis Webportal: The RT2 Profiler PCR Array Data Analysis Webportal analyzes CT values to calculate changes in gene expression. For the RT2 Profiler PCR Array Data Analysis Webportal to correctly read/import your data, the CT values must be exported from your qPCR instrument and then formatted into a new Excel spreadsheet. The RT2 Profiler PCR Array Data Analysis Webportal only accepts Excel spreadsheets for analysis. All of the plates/wells must be copied into a single worksheet. Please use the templates below as guides. 1. If you are using a catalogued PCR Array where 1 sample was tested on a 96 well plate, 100 well ring or 384 well plate (the 1x384 layout), the Raw CT values should be in a single column that corresponds to the correct well location on the PCR Array. A blank spreadsheet is available at: http://www.sabiosciences.com/pcr/template.xls 2. If you are starting with a 384 well plate which is in the 4x96 layout, then you will need to separate each sample’s data into a separate column, by using the 384-Well Format E/G Data Analysis Patch tool located at http://www.sabiosciences.com/pcrarraydataanalysis.php 3. If you are using a custom PCR Array, the Raw CT values for each sample should be in a single column that corresponds to the correct well location (column A) on the PCR Array. The gene tested in each well must be supplied by the user in column B. A sample spreadsheet is available at: http://pcrdataanalysis.sabiosciences.com/pcr/templates/customarray_template.xls 4. If you are analyzing data from qPCR primer, primer/probe or Taqman gene expression experiments, the Raw CT values for each sample should be in a single column that corresponds to the correct well location (column A) on the PCR Array. The gene tested in each well must be supplied by the user in column B. A sample spreadsheet is available at: http://pcrdataanalysis.sabiosciences.com/pcr/templates/assay_template.xls Upload and annotate the data: 1. Choose the experiment that was performed: Standard/Cataloged RT² Profiler PCR Array, Custom RT² PCR Array or individual assays a. If you selected Standard RT² PCR Array then Enter the PCR Array Pathway Number from the drop-down list. Note: The RT² Profiler PCR Array System was upgraded to Version 4.0 on May 25, 2012. The Version 4.0 arrays added a “Z” to their pathway numbers. Therefore, a Version 3.0 PCR Array has the following number PAXX-###, while a Version 4.0 PCR Array has the following number PAXX-###Z. The XX denotes the species (HS = Human, MM = Mouse, RN = Rat, …) the ### denotes the pathway/gene list. The letters (A, C, D, E, F, H, G, R) that follow the ### or the “Z” can be ignored, they represent the plate format. b. If you selected Custom RT² PCR Array then Enter the Custom Array ID (ex. CAPX####) in the text field. c. If your experiment used individual primer assays then select Single or Multi-Gene qPCR Assays. 2. Browse and select the MS Excel file that you prepared in the above section (Formatting data for the Data Analysis Webportal) that contains all of your PCR data with a maximum number of 100 samples. Click Upload. Page 2 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu
  3. 3. Quick Reference: RT2 Profiler PCR Array Data Analysis Setup and quality check the uploaded data: 1. The uploaded raw CT data is displayed in the window underneath the Analysis Setup tab. 2. In the Basic Setup section, assign samples to different groups. At least two groups are needed, where one of those groups must be the control group. Click Update when finished. You may exclude samples from the analysis by selecting Exclude on the drop down menu. a. Review the Data QC section to assess each groups' PCR reproducibility, reverse transcription efficiency, and the presence of genomic DNA contamination. b. The Select Housekeeping Genes section allows you to remove or add preferred housekeeping genes for data normalization by clicking the appropriate checkboxes. Click Update when finished. Note: The user must click on Perform Normalization before proceeding with the analysis. c. Review the Data Overview section to see each group's distribution of threshold cycle values and the average of the raw data in each group. Analysis page: 1. See the Average CT, 2^(- CT), Fold Change, p-value, and Fold Regulation sections for the results processed by the software from your data. The Fold Change and p-value results are used by the software in subsequent graphical analyses. Note: The fold change (fold difference) is calculated by the equation 2(-∆∆CT). For the fold regulation, the software transforms fold change values less than 1 (meaning that the gene is down regulated) by returning the negative inverse. The values of the numbers are the identical; the software has only changed the representation of the value. For example if ABL1 has a fold change value of 0.31, this is equivalent ABL1 having a fold regulation of -3.2 fold. (ABL1 expression is decreased 3.2 fold). Plots and Charts: 1. Heat Map a. Define Groups to be compared and whether to report the fold-change results as a log transformation or not (default recommended). Click Update once changes have been made. Click Export Data to download the results as an Excel file. b. To save the figure: 1. Using Windows: Right-click on the figure and save the image/picture 2. Using OS X: Hold down the Control key & Click and save the image/picture 2. Scatter Plot a. Define Groups to be compared. Choose the fold-change boundary (and p-value for Volcano Plot) of interest. Click Update. b. Click on symbols to identify gene and fold-change (and p-value for Volcano Plot); mouse over table entries to point to symbol on plot. c. Click check boxes to remove genes from or add genes to plot. Click Update. d. Click Export Data to download the results as an Excel file. e. To save the figure: 1. Using Windows: Right-click on the figure and save the image/picture 2. Using OS X: Hold down the Control key & Click and save the image/picture 3. Volcano Plot a. Define Groups to be compared. Choose the fold-change boundary (and p-value for Volcano Plot) of interest. Click Update. b. Click on symbols to identify gene and fold-change (and p-value for Volcano Plot); mouse over table entries to point to symbol on plot. c. Click check boxes to remove genes from or add genes to plot. Click Update. Page 3 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu
  4. 4. Quick Reference: RT2 Profiler PCR Array Data Analysis d. Click Export Data to download the results as an Excel file. e. To save the figure: 1. Using Windows: Right-click on the figure and save the image/picture 2. Using OS X: Hold down the Control key & Click and save the image/picture 4. Clustergram a. Sort samples by "Array" or by "Group". b. Select "Join Type". The default of "Average" is recommended. c. Cluster in "1-D" by genes only or in "2-D" by genes and samples. d. Color code the graph by "Genes", "Samples" or "Entire Dataset". The default of "Genes" is recommended. e. Choose whether or not to display the gene symbols and array or group names. f. Click check boxes to remove genes from or add genes to plot. g. Click Update. h. To save the figure: 1. Using Windows: Right-click on the figure and save the image/picture 2. Using OS X: Hold down the Control key & Click and save the image/picture 5. Multigroup Plot a. Click check boxes to add genes to or remove genes from plots. b. Choose results to plot on the y-axis, either "AVG Delta CT", "2^Delta CT", or "Fold Change". c. Click Update. d. Review either the line plot (above) or the column chart (below). e. To save the figure: 1. Using Windows: Right-click on the figure and save the image/picture 2. Using OS X: Hold down the Control key & Click and save the image/picture Export the Results: 1. Click Export Data to download a MS Excel file containing all raw and processed data from the "Readout" and "Analysis Result" sections. Note: The RT2 Profiler PCR Array Data Analysis Webportal does not save any results. Please ensure that you have exported or copied any figures or tables from each session. What’s Next The Data Analysis software delivers a list of expression changes in the samples from the supplied data. In order to assist in further analysis, the software utilizes the latest bioinformatics tools to analyze the data. Please use each sub-menu to examine the results. 1. Gene Expression a. This tool will help define a panel of genes based of experiment’s results. The tool is designed to deliver a list of gene expression assays that would allow the user to follow-up the results of the analyzed experiment. 2. miRNA Regulation a. This tool will identify candidate miRNA regulators in your experimental system. The tool delivers a list of miRNAs that could be targeting the genes that had observed changes in expression in your selected samples. RT2 Profiler PCR Arrays, RT2 PCR Assays, and RT2 SYBR Mastermixes are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Page 4 of 4 QIAGEN GmbH  QIAGEN Str. 1  40724 Hilden  Germany  Commercial Register Düsseldorf (HRB 45822) Managing Directors: Peer M. Schatz, Roland Sackers, Bernd Uder, Dr. Joachim Schorr, Douglas Liu

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